Exposed proteins of the Schistosoma japonicum tegument

The ability of the mammalian blood fluke Schistosoma japonicum to survive in the inhospitable environment of the mammalian bloodstream can be attributed, at least in part, to its host-exposed outer surface, called the tegument. The tegument is a dynamic organ and is involved in nutrition, immune evasion and modulation, excretion, osmoregulation and signal transduction. Given its importance for parasite survival, proteins exposed to the host at the surface of the tegument are ideal targets for the development of vaccines and drugs. By biotinylating live adult worms and using a combination of OFFGEL electrophoresis and tandem mass spectrometry 54 proteins were identified as putatively host-exposed in S. japonicum. These included glucose transport proteins, an amino permease, a leucine aminopeptidase and a range of transporters, heat shock proteins and novel immune-active proteins. Members of the tetraspanin protein family and a homologue of Sm 29, a tegument membrane protein from Schistosoma mansoni, both effective vaccine antigens in S. mansoni, were also identified. The fate of labelled surface proteins was monitored over time using electron microscopy and revealed that biotinylated proteins were rapidly internalised from the surface of the tegument and trafficked into the cytoplasmic bridges that connect the distal cytoplasm of the tegument to the underlying cell bodies. The results reported herein dramatically increase the number of S. japonicum proteins known to be exposed to the host and, hence, those of interest as therapeutic targets. The ability of the parasite to rapidly internalise proteins at its surface has implications for the development of vaccines and may explain how these parasites are able to avoid the host immune system for long periods of time.     

Schistosoma mansoni Annexin 2: Molecular characterization and immunolocalization

We here describe the cloning and characterization of the Schistosoma mansoni Annexin 2, previously identified in the tegument by proteomic studies, and as an up-regulated gene in schistosomulum stage by microarray data. In silico analysis predicts a conserved core containing four repeat domains of Annexin (ANX) and a variable N-terminal region similar to that described for mammalian isoforms. Real-time RT-PCR and Western blot analysis determined that S. mansoni Annexin 2 is significantly up-regulated in the transition from free-living cercaria to schistosomulum and adult worm parasitic stages. Immunolocalization experiments and tegument membrane preparations confirmed Annexin 2 as a protein mainly localized in the tegument of schistosomula and adult worms. Furthermore, it binds to the tegument surface membranes in a calcium-dependent manner. These results suggest that S. mansoni Annexin 2 is closely associated to the tegument arrangement, being a potential target for immune intervention.     

Development of Schistosoma mansoni in the laboratory rat analyzed by light and confocal laser scanning microscopy

The kinetic of maturation (schistogram) of Schistosoma mansoni worms grown in laboratory rats was studied by light and confocal laser scanning microscopy. Infected rats with the BH strain were weekly euthanized 3-9 weeks pi. Recovered flukes stained with hydrochloric carmine were preserved as whole-mounts and analyzed by confocal and brightfield microscopy. Worms displayed varying degrees of maturation of the reproductive system at weeks 3-6. Male worms showed complete maturation of the reproductive system at week 6, while female worms completed their maturation at week 7. Males presented few tubercles in tegument in all weeks. Despite the presence of a developing embryo within the ootype, no uterine egg was found. The schistogram in rats follows a pattern similar to that observed in mice hosts.     

Schistosoma mansoni host-exposed surface antigens characterized by sera and recombinant antibodies from schistosomiasis-resistant rats

Antibodies from Schistosoma mansoni-infected rats, unlike mice, show a higher titer for schistosome apical tegumental antigens compared with non-apical membrane antigens. These antibodies bind to the surface of living lung-stage worms and to formaldehyde-fixed adult worms. We produced a single-chain antibody Fv domain (scFv) phage library displaying the antibody repertoire of rats highly immune to schistosome infection and we selected for scFvs that recognize the host-exposed surface of worms. Five unique rat scFvs (Teg1, Teg4, Teg5, Teg20 and Teg37) were obtained which recognize schistosome surface epitopes. Each of the scFvs recognizes the surface of living schistosomula and lung-stage schistosomules and/or the surface of formaldehyde-fixed adult worms. None of these scFvs reproducibly stained living adult worms. This suggests that a change occurs during the transition from lung schistosomules to 4-week adults such that at least some surface antigens, although remaining on the surface in living adult worms, can no longer be immunologically stained. Teg1 and Teg4 scFvs both recognize specific bands on Western blots. No bands were observed for the other three scFvs, suggesting that these scFvs may recognize non-protein or conformationally-dependent epitopes. Teg1 was unambiguously identified as recognizing the S. mansoni tetraspanin antigen, SmTSP-2, within the large extracellular domain. Teg4 recognizes a 35 kDa band tentatively identified as Sm29 by proteomic analysis. These scFvs can now be used to characterize schistosome epitopes at the host-parasite interface, to target worms in vivo, and to study the mechanisms by which these worms naturally evade immune damage to the tegument within permissive hosts.     

Schistosoma mansoni: changes in the outer membrane of the tegument during development from cercaria to adult worm

Hockley D. J. and McLaren D. J. 1973. Schistosoma mansoni: changes in the outer membrane of the tegument during development from cercaria to adult worm. International Journal for Parasitology3: 13–25. The tegumental outer membrane of the cercaria is trilaminate: the adult worm, however, has a seven-layered membrane. Formation of the heptalaminate membrane commences immediately after the cercaria has penetrated the vertebrate host: multilaminate membrane-bounded vacuoles are passed from subtegumental cells into the tegument where they enlarge, join to the outer membrane and open to the exterior. The heptalaminate limiting membrane of the vacuole thus becomes the outer membrane of the tegument. At the same time the original trilaminate tegumental membrane is formed into microvilli which are cast off and thus the cercarial outer membrane is lost. Schistosomula usually have a heptalaminate outer membrane within three hours of penetration. After this time the large vacuoles are replaced by smaller membraneous bodies which presumably contribute to the outer membrane during growth of the schistosomulum. The membraneous bodies are also present in the tegument of the adult worms and there is some evidence that the outer membrane is continually renewed.     

Effect of amphotericin B on the infection success of Schistosoma mansoni in Biomphalaria glabrata

In the present study, we examined the effect of amphotericin B on larval stages (miracidia and primary sporocyst) of the helminth Schistosoma mansoni, the causative agent of human schistosomiasis. Amphotericin B (AmB) is a polyene macrolide that disturbs the function of the cell membrane; it is widely used as prophylactic antimycotic agent in in vitro culture. We show for the first time that S. mansoni miracidia infectivity is considerably reduced after AmB treatment. Moreover we demonstrate that AmB does not affect the development, growth, viability, and behavior of miracidia and primary sporocysts. Our data indicate that AmB effects on S. mansoni sporocyst prevalence are linked to the oxidative properties of AmB. These may alter the capacity of sporocysts to respond to the oxidative stress generated by the snail immune defence system.     

The Effect ofBacillus thuringiensisM-exotoxin on Trematode Cercariae

The effect ofBacillus thuringiensis israelensiseluate containing water-soluble exotoxin (M-exotoxin) was observed by its use on cercariae of seven trematode species. To the most sensitive species to the toxic effect of the mentioned toxin belonged schistosome furcocercariae (human speciesSchistosoma mansoniand avian parasiteTrichobilharzia szidati). Under the influence of the toxin, surface syncytial structure (tegument) was separated from underlying tissues, with subsequent disintegration of internal organs connected with disruption of acetabular glands and release of their proteolytic content.     

The cellular distribution and stage-specific expression of two dynein light chains from the human blood fluke Schistosoma japonicum

Schistosomes are pathogenic helminth parasites of human portal veins. Their body wall is a highly active syncytial tegument involved in an array of host interactions. The cytoskeletal organization and dynamics of this syncytium are poorly understood, but predominant motor components are the LC8 class of cytoplasmic dynein light chains (DLC). Four LC8 members occur in schistosomes, two of which are expressed in the tegument. Here, we describe the cytoplasmic distribution, stage-specific expression and cellular location of two diverse LC8 molecules of Schistosoma japonicum. SjDLC1 was detected in surface-membrane specific extracts of adult worms and was shown by quantitative immuno-electron microscopy to predominate along heptalaminate membranes of the worm surface. SjDLC3 also occurs in the tegument, but was shown to be present in basal layers of the tegument and did not preferentially co-localize with particular membrane components. SjDLC3 was also detected in the gastrodermis. SjDLC1 is expressed only in mammalian-parasitic stages, whereas SjDLC3 is expressed throughout the life-cycle. The data suggest that SjDLC1 is preferentially located to the host-interactive distal parasite membrane, and plays a role in surface membrane dynamics, while SjDLC3 is a ubiquitous motor component of schistosome epithelia of all stages.     

Transmission electron microscopic observations on lipid release in Schistosoma mansoni maintained in vitro

Transmission electron microscopy was done on Schistosoma mansoni adult males and females unpaired immediately following recovery from mice and on worms maintained in Earle's balanced salt solution (EBSS) containing 0.1% glucose and 0.5% lactalbumin hydrolysate for 1.0 h at 37°C. Males incubated for 0.5 and 1.0 h released lipids from non-tuberculate and tuberculate areas of the dorsal tegument. Lipid release was not seen in females. Transmission electron microscopy revealed no apparent damage in worms maintained in BBSS for 1.0 h. Our observations suggest that structures described as “spheres” (Weisberg, Carlisle &; Bentley, 1983) are lipid droplets.     

Functional Mapping of Protein Kinase A Reveals Its Importance in Adult Schistosoma mansoni Motor Activity

Cyclic AMP (cAMP)-dependent protein kinase/protein kinase A (PKA) is the major transducer of cAMP signalling in eukaryotic cells. Here, using laser scanning confocal microscopy and ‘smart’ anti-phospho PKA antibodies that exclusively detect activated PKA, we provide a detailed in situ analysis of PKA signalling in intact adult Schistosoma mansoni, a causative agent of debilitating human intestinal schistosomiasis. In both adult male and female worms, activated PKA was consistently found associated with the tegument, oral and ventral suckers, oesophagus and somatic musculature. In addition, the seminal vesicle and gynaecophoric canal muscles of the male displayed activated PKA whereas in female worms activated PKA localized to the ootype wall, the ovary, and the uterus particularly around eggs during expulsion. Exposure of live worms to the PKA activator forskolin (50 µM) resulted in striking PKA activation in the central and peripheral nervous system including at nerve endings at/near the tegument surface. Such neuronal PKA activation was also observed without forskolin treatment, but only in a single batch of worms. In addition, PKA activation within the central and peripheral nervous systems visibly increased within 15 min of worm-pair separation when compared to that observed in closely coupled worm pairs. Finally, exposure of adult worms to forskolin induced hyperkinesias in a time and dose dependent manner with 100 µM forskolin significantly increasing the frequency of gross worm movements to 5.3 times that of control worms (P≤0.001). Collectively these data are consistent with PKA playing a central part in motor activity and neuronal communication, and possibly interplay between these two systems in S. mansoni. This study, the first to localize a protein kinase when exclusively in an activated state in adult S. mansoni, provides valuable insight into the intricacies of functional protein kinase signalling in the context of whole schistosome physiology.     

Schistosoma mansoni: Ultrastructural damages due to complement on schistosomula in vitro

The ultrastructural damage induced by complement in vitro on the schistosomula of Schistosoma mansoni was studied using transmission and scanning electron microscopy. The sequence of events which leads to the killing of the schistosomula is as follows: (a) the lytic activity starts at the anterior end of the schistosomula; (b) lesions progress simultaneously in two distinct directions: from the anterior to the posterior end, and from the outer membrane to the muscle layer; (c) “bubbles” appear around parasite which might correspond to increased membrane permeability; (d) the lytic activity of the late complement components occur in the syncytium; (e) the schistosomula lose their tegument completely, exposing the muscle layer. These findings and our previous work suggest that the activation of the alternate pathway and late complement components is sufficient, without antibodies or cells, to kill schistosomula in vitro.     

Schistosoma mansoni: human skin ceramides are a chemical cue for host recognition of cercariae

Schistosoma mansoni cercariae recognize the human host with a sequence of behavioral responses particularly to chemical host cues. After attaching to the skin surface, cercariae are stimulated by so far unknown skin components to hold enduring contact with the skin and to start creeping towards entry sites. We studied the chemical stimulus of human skin for the cercarial enduring contact response by fractionation of human and pig skin surface extracts and offering the fractions to the cercariae via membrane filters. Enduring contact was stimulated exclusively by ceramides, specific lipids of the uppermost skin layers. This chemical cue differs from the 6 chemical host signals used by S. mansoni cercariae in other behavioral steps of host invasion, and thus underlines the specialization of S. mansoni cercariae particularly in chemical host signals. All together, the enduring contact response of the cercariae is, like the other phases of host invasion, well adapted to the chemical properties of human skin.     

Cytochemical and biochemical observations on the digestive tracts of digenetic trematodes. 8. Acid phosphatase activity in Schistosoma mansoni and Schistosomatium douthitti

At the light microscope level, nonspecific acid phosphatase (AcPase) (EC 3.1.3.2) and N-acetyl glucosaminidase (NAGase) (EC 3.2.1.29) activities are in the esophageal gland cells of Schistosoma mansoni and Schistosomatium douthitti and in the gastrodermis of S. mansoni. The gastrodermis of S. douthitti is negative for these two enzymes. At the electron microscope level, AcPase activity in the esophageal gland cells of both species is observed in cytoplasmic vesicles. In S. mansoni, AcPase activity is also observed associated with the infoldings of the basal plasma membranes of the esophagus and the gastrodermis. It is hypothesized that this enzyme(s) is involved with membrane transport. AcPase activity is also associated with “droplets” and vesicles in the gastrodermis of S. mansoni. It is believed that the digestion of foodstuffs in both species occurs extracellularly.     

Schistosoma mansoni and Schistosoma haematobium: Differences in development

Growth and maturation of the Puerto Rico strain of Schistosoma mansoni in mice and the Ghana strain of Schistosoma haematobium in hamsters were compared beginning 19 days after infection. In S. mansoni, optimum development was determined, with copulation first observed on Day 25, egg shell protein formation observed on Day 28, and oviposition occurring on Day 30. In infections of S. haematobium, copulation first occurred on Day 29. Egg shell proteins were first formed on Day 45, and egg production occurred on Day 60. Examination of unisexual and bisexual infections showed that maturation of the vitellaria can be more easily assessed by autofluorescence of the protein globules than by the traditional diazonium salt stains. Autofluorescence of living worms with mature vitellaria allows subsequent examination by electron microscopy, and therefore permits evaluation at a subcellular level.     

Intermediate host specificity in Schistosoma mansoni

Miracidia of Schistosoma mansoni penetrate into many kinds of snails, but development of normal sporocysts takes place only in certain species of Biomphalaria. Different populations of this snail vary greatly in laboratory infection rates with S. mansoni originating from diverse geographic localities. Cross-exposure experiments show that compatibility factors exist in both snails and parasites. Susceptibility of stocks of Biomphalaria to particular strains of S. mansoni is genetically determined and may be modified by selection in the laboratory. In a compatible snail, the sporocyst develops without host tissue reaction; in incompatible snails the early larvae are rapidly surrounded by amebocytes and fibroblasts, and destroyed. This reaction resembles the generalized host cellular response elicited by any foreign body. An individual snail exposed to many miracidia may have both developing and encapsulated sporocysts side by side within its tissues. The weight of current evidence suggests that elicitation or absence of this cellular response resides in the recognition or nonrecognition of the sporocyst as a foreign body. The sporocyst tegument surface, which forms within a few hours after miracidial penetration, may have a molecular conformation identical with that of the snail, or may be able to bind specific host molecules, so that detection and subsequent encapsulation by host cells are averted. Presuming genetic determination of the sporocyst surface structure and of the host cell detection capability, differing infection rates would result from the particular frequencies of relevant genes in the populations concerned.     

Schistosoma spp.: Isolation of microtubule associated proteins in the tegument and the definition of dynein light chains components

Schistosomes are parasitic blood flukes that reside in human mesenteric veins or urinary bladder veins, depending on species of the parasite. The syncytial tegument of these parasites represents a dynamic interface that regulates nutritional and immunological interactions between the parasite and the host. It is known that the components for biogenesis and maintenance of the tegument are supplied via vesicles from the nucleated cell bodies beneath the syncytium and muscle layer. To investigate the common motor components of vesicular transport in the tegument of schistomes, we extracted Schistosoma mansoni tegumental microtubule associated proteins utilizing detergent/high-salt procedure and raised antiserum against these proteins. The antiserum was applied to screen Schistosoma haematobium λgt11 expression library and some of the isolated clones were sequenced. Blast search for the sequences against NCBI database identified clones that are dynein light chains and myosin genes. Further analysis of schistosome dynein genes in the databases identified three families of dynein light chains (Dlcs). The Tctex family protein sequences are significantly different from the mammalian homologs and, therefore, offer a potential vaccine/drug target against schistosomes.     

Schistosoma mansoni: effect of a protein-free host diet on tegument.

The effect of a protein deficiency in the host's diet on the tegument of Schistosoma mansoni is described. Both the infected and the uninfected hamsters, fed on the diet, were stunted in growth; but the effect of the diet was more pronounced on the infected hamsters. The parasites recovered from both the liver and the mesenteric veins of animals fed on the diet from the time of infection were also retarded in growth. The tegument of both groups of parasites were reduced in height as compared with the tegument of control worms. The worms recovered from the liver of the hamsters were less adversely affected than the worms recovered from the mesenteric veins, in the sense that the tegument did not show any sign of degeneration, as was found in the latter group of parasites. In the worms from the mesenteric veins, the external plasma membrane was approximately half the thickness of the external plasma membrane of control worms. The invaginations of the external plasma membrane of experimental worms penetrated deeply into the tegument and in most instances almost reached the basal plasma membrane. Prolonged feeding of the hosts on the experimental diet resulted in the disintegration of the tegument in localised areas of the body. There was no adverse effect on adult worms of an established infection after the hosts were transferred to the protein-free diet for up to 3 wk. The ability of the tegument to regenerate after transferring the hosts from the experimental diet to normal laboratory diet (control diet) was demonstrated.     

Enzymatic shaving of the tegument surface of live schistosomes for proteomic analysis: a rational approach to select vaccine candidates

Background

The membrane-associated and membrane-spanning constituents of the Schistosoma mansoni tegument surface, the parasite''s principal interface with the host bloodstream, have recently been characterized using proteomic techniques. Biotinylation of live worms using membrane-impermeant probes revealed that only a small subset of the proteins was accessible to the reagents. Their position within the multilayered architecture of the surface has not been ascertained.

Methodology/Principal Findings

An enzymatic shaving approach on live worms has now been used to release the most accessible components, for analysis by MS/MS. Treatment with trypsin, or phosphatidylinositol-specific phospholipase C (PiPLC), only minimally impaired membrane integrity. PiPLC-enriched proteins were distinguished from those released in parasite vomitus or by handling damage, using isobaric tagging. Trypsin released five membrane proteins, Sm200, Sm25 and three annexins, plus host CD44 and the complement factors C3 and C4. Nutrient transporters and ion channels were absent from the trypsin fraction, suggesting a deeper location in the surface complex; surprisingly, two BAR-domain containing proteins were released. Seven parasite and two host proteins were enriched by PiPLC treatment, the vaccine candidate Sm29 being the most prominent along with two orthologues of human CD59, potentially inhibitors of complement fixation. The enzymes carbonic anhydrase and APD-ribosyl cyclase were also enriched, plus Sm200 and alkaline phosphatase. Host GPI-anchored proteins CD48 and CD90, suggest ‘surface painting’ during worm peregrination in the portal system.

Conclusions/Significance

Our findings suggest that the membranocalyx secreted over the tegument surface is not the inert barrier previously proposed, some tegument proteins being externally accessible to enzymes and thus potentially located within it. Furthermore, the detection of C3 and C4 indicates that the complement cascade is initiated, while two CD59 orthologues suggest a potential mechanism for its inhibition. The detection of several host proteins is a testimonial to the acquisitive properties of the tegument surface. The exposed parasite proteins could represent novel vaccine candidates for combating this neglected disease.     

Schistosoma mansoni: axenic culture of daughter sporocysts

Daughter sporocysts of Schistosoma mansoni were cultured axenically for up to 13 days in media conditioned with Aedes albopictus tissue cultures. Sporocysts increased in length, processes appeared on the tegument, and small embryos developed. Two media, differing in ionic balance and source of amino nitrogen, were compared. No development occurred in either medium when freshly prepared.